Methods for isolation, use and analysis of ferritin

ABSTRACT

This invention provides methods of isolating ferritin from plant and animal material. The isolated ferritin can be administered to humans or animals in need of iron, and can be used to treat or supplement iron deficiency. The isolated ferritin can be used in industrial applications, such as increasing the iron content in heat-processed food or beverages. The methods of the invention also include quantitation of iron derived from plant or animal ferritin.

CROSS-REFERENCE TO RELATED APPLICATIONS

The present application is a Continuation of U.S. patent applicationSer. No. 13/908,635, filed on Jun. 3, 2013, titled “Methods forIsolation, Use and Analysis of Ferritin,” which is a Divisional of U.S.patent application Ser. No. 13/730,618, filed on Dec. 28, 2012, now U.S.Pat. No. 8,476,061 titled “Methods for Isolation, Use and Analysis ofFerritin,” which claims the benefit of U.S. Provisional PatentApplication No. 61/581,780, filed on Dec. 30, 2011, titled “Methods forPurification, Use and Analysis of Ferritin,” and U.S. Provisional PatentApplication No. 61/581,809, filed on Dec. 30, 2011, titled “Methods forIsolation, Use and Quantitation of Ferritin,” the contents of which areincorporated in this disclosure by reference in their entirety.

BACKGROUND

In humans and other animals, iron is essential for the implementationand maintenance of many vital cellular functions and biosyntheticprocesses, including oxygen transport, aerobic cellular activity,intracellular electron transport, and integral enzymatic reactionswithin body tissue. Iron deficiency is the most common nutritionaldeficiency worldwide, affecting 30 million people in both developed anddeveloping countries. Iron deficiency has many repercussions includingdiminishing growth and learning in children.

The majority of stored iron in body tissues is contained in ferritin.Ferritin is an intracellular, protein-iron complex, formed fromself-assembling subunits. The protein cage can reversibly form iron intoa caged biomineral, Fe₂O₃·H₂O, in plants, animals, and bacteria. Ironoxy-biominerals inside the protein nanocages are iron concentrates forprotein synthesis, and Fe(II)/oxygen/peroxide traps (Fenton chemistryreactants) for antioxidant protection. The iron contained in ferritin isconcentrated 100 billion times above the solubility of ferric ion in anontoxic, accessible form. Ferritin protein subunits, four a-helixbundles, contain a catalytic center that converts two Fe(II) atoms to anFe(III)-oxo bridged dimer intermediate in mineralization. The twoclasses of ferritins are: i) maxi-ferritins, 24-polypeptide, 4-bundlesubunit assemblies found in animals, plants, and bacteria; and ii)mini-ferritins (also called Dps proteins), 12-polypeptide, 4-bundlesubunit assemblies in archaea and bacteria.

In animals, ferritin is mainly present in tissues, especially in theliver, kidney, spleen and bone marrow erythroid cells where it serves asan iron reserve for the production of hemoglobin. A small fraction offerritin is in the serum and contributes little to overall iron storage,but is used clinically as a reporter of iron levels in an animal.Ferritins occur in animals as approximately 25 distinct isoformsdepending on their proportions of the two primary subtypes of ferritins,H or L. These distinct subtypes differ in their tissue distribution,rates and mechanisms of iron oxidation, core formation and physiologicaliron turnover.

Ferritin derived from plants and animals can be used as a dietary sourcefor humans and other animals. Ferritin, which survives digestion largelyintact, is more efficiently absorbed by the intestine than any otherdietary iron source or iron supplement, because of the large amount ofiron per ferritin molecule. Ferritin also survives treatment with highheat. The ferritin protein makes ferritin iron a naturally entericcoated, slow release, efficiently absorbed iron source. As such,ferritin can used to supplement iron in animals in need of increasediron in their diet.

Currently, there is a need for methods to isolate plant and animalferritin from a low-cost, readily available source, which can beadministered to a subject in an amount to treat an iron deficiencydisorder or prevent iron deficiency. There is also a need for methods todetermine the amount of iron derived from animal and plant ferritin.

SUMMARY

The invention describes a method for isolating ferritin from a legume ora legume processing stream by the steps of (a) separation of the legumeor the legume processing stream into soluble and insoluble legumefractions; (b) addition of a neutral saline buffer to the insolublelegume fraction to make an insoluble legume solution; (c) clarificationof the insoluble legume solution of step (b) into soluble and insolublelegume solution fractions; (d) enzymatic treatment of the clarifiedsoluble legume solution of step (c); (e) fractionation of theenzymatically treated, clarified soluble legume solution of step (d) toremove non-ferritin components; and (f) concentration of isolatedferritin from the fractionated soluble legume solution of step (e).

The invention also describes a method for isolating ferritin from legumeor a legume processing stream by the steps of (a) separation of thelegume or the legume processing stream into soluble and insoluble legumefractions; (b) addition of a neutral saline buffer to the insolublelegume fraction to make an insoluble legume solution; (c) clarificationof the insoluble legume solution of step (b) into soluble and insolublelegume solution fractions; (d) enzymatic treatment of the clarifiedsoluble legume solution of step (c); (e) fractionation of theenzymatically treated, clarified soluble legume solution of step (d) toremove non-ferritin components; and (f) concentration of isolatedferritin from the fractionated soluble legume solution of step (e).

Also described herein is a method to isolate ferritin from plantmaterial by the steps of: (a) separation of the plant material into asoluble and insoluble fraction; (b) removal of non-ferritin componentsfrom the soluble fraction of step (a), thereby isolating ferritin; and(c) concentration of the isolated ferritin from step (b). The plantmaterial can be from legumes, such as soybeans.

It is contemplated that the plant material can consist of the entireplant, or it can be a part of a plant, for example one or more of aseed, a bean, a stem, a fruit, a leaf, a root, and a flower. The plantmaterial can be material from the waste stream from processing oflegumes.

In one aspect, the step of soluble and insoluble separation can be doneby mechanical separation.

It is contemplated that the step of removal of non-ferritin componentsis done by enzymatic treatment of the soluble fraction. Theenzymatically treated non-ferritin components can be removed. The enzymeused to remove the non-ferritin components can be one or more of aglycosidase enzyme.

The isolated ferritin can be concentrated by, for example, drying thesoluble fraction, or by ultrafiltration.

Additional steps can be added to the method of isolating ferritindescribed above, such as fractionation of the soluble fraction eitherbefore or after the non-ferritin components are removed. In addition,the soluble fraction can be treated with a solvent such as, for example,an organic solvent.

Also described is a method to isolate ferritin from animal material bythe steps of: (a) separation of the animal material into soluble andinsoluble fractions; (b) heat-denaturation of the soluble fraction; (d)removal of carbohydrates from the heat-denatured soluble fraction byenzyme digestion; and (c) concentration of the enzymatically treated,heat-denatured soluble fraction. The animal material can be animaltissues such as, for example, the liver, kidney, spleen or bone marrowfrom an animal.

The step of soluble and insoluble separation can be mechanicalseparation. The enzyme used in step (d) can be one or more of aglycosidase enzyme. It is contemplated that the heat-denaturation isperformed at temperatures below 80° C. Concentration of the isolatedferritin can be accomplished by drying the soluble fraction with lowheat, spray drying, or ultrafiltration.

Additional steps can be added to the method of isolating ferritin fromanimal material, including size fractionation after enzymatic treatmentof the soluble fraction. Another step that can be added is treatment ofthe soluble fraction with a solvent such as, for example, an organicsolvent.

The invention is also directed towards the isolated ferritin obtained bythe methods described herein.

The invention also describes a method for determining the amount offerritin iron in a sample wherein the sample has a known weightpercentage of a ferritin iron source having the following steps: (a)measuring the total iron in the sample per unit weight of the sample;(b) preparing a soluble extract of a known weight per volume of thesample from step (a) to make a soluble sample extract; (c) determiningthe total iron per unit weight of the soluble sample extract of thesample; (d) determining the ferritin protein per unit weight in thesoluble sample extract, using quantitative immunological analysis; and(e) calculating the amount of ferritin iron in the sample using theamount of the total iron from step (c) divided by the amount of ferritinprotein from step (d), multiplied by the amount of total iron in thesample from step (a) multiplied by a correction factor, therebydetermining the amount of ferritin iron in the sample. For a sample oflegumes, the correction factor is 0.75.

The ferritin iron source used in this method can be material from aplant or an animal. The sample containing ferritin iron can be derivedfrom animal ferritin in the normal tissue or hyperferritinemic serum ofa subject.

It is contemplated that the quantitative immunological analysis can bedone by any means, for example, quantitative immunoblot, Western blotanalysis, and quantitative capillary immunoelectrophoresis.

The invention also describes the use or treatment of a condition causedby iron-deficiency in an organism in need thereof by the use oradministration of an effective amount of isolated ferritin obtained bythe methods herein.

The organism in need of treatment could be any organism, for example, aplant or an animal such as a mammal, including a human.

The invention also describes a method of increasing the iron content ofheat-processed substance by the addition of isolated ferritin obtainedfrom the methods described herein. The isolated ferritin can be added tothe substance before the substance is heat-processed. In one embodiment,the heat-processed substance is food. In another embodiment, theheat-processed substance is a beverage.

BRIEF DESCRIPTION OF THE DRAWINGS

These and other features, aspects, and advantages of the presentinvention will become better understood with regard to the followingdescription, appended claims, and accompanying drawings.

FIG. 1 depicts a graph demonstrating the enzymatic digestion of starchin ferritin extracts over time.

DETAILED DESCRIPTION Definitions

As used herein, the following terms and variations thereof have themeanings given below, unless a different meaning is clearly intended bythe context in which such term is used.

The terms “a,” “an,” and “the” and similar referents used herein are tobe construed to cover both the singular and the plural unless theirusage in context indicates otherwise.

The term “buffer” or “buffered solution” refers to a mixture of acid andbase which, when present in a solution, reduces or modulates changes inpH that would otherwise occur in the solution when an acid or base isadded,

As used herein, the term “comprise” and variations of the term, such as“comprising” and “comprises,” are not intended to exclude otheradditives, components, integers or steps.

A “correction factor” as used herein refers to a number used incalculations of the amount of ferritin iron in a sample. The correctionfactor is a number that must be multiplied by the minimum percentage offerritin iron in the original ferritin iron source, because the sourceof ferritin iron may contain iron in addition to ferritin iron. As such,the correction factor must be used so that an error in calculation doesnot occur. As an example, in legumes, a minimum of 75% of the iron isferritin iron, so a correction factor of 0.75 would be used with legumesources of ferritin iron. However, other sources of ferritin iron willhave different correction factors.

As used in this specification, the term “ferritin” means the proteinwith caged iron oxide mineral that confers highly efficient intestinaliron absorption in living organisms and has biological and/or chemicalactivity and structure the same as, or substantially similar to, anatural, iron-containing ferritin. As such, ferritin includes anaturally occurring ferritin protein with iron mineral or a recombinant,iron mineral-reconstituted ferritin protein, comprising 12 or 24ferritin subunits, wherein the subunits associate to form a sphericalnanocage. Natural ferritins include human ferritin, ferritin from otheranimals (ferritin derived from horse spleen or bullfrogs, for example),plant ferritin (derived from soybeans, and any other legume or legumeprocess stream for example), microbial ferritins: mycoferritin (derivedfrom fungi), or bacterial ferritin (bacterioferritin) or archaealferritin. Ferritin protein includes recombinant ferritin expressed bygenetically-transformed microorganisms such as E. coli, and otherbacteria and yeasts. Ferritin expressed by genetically-transformed orrecombinant microorganisms can have an amino acid sequence identical oranalogous to a natural ferritin. The term ferritin protein can includeprotein cages consisting of one or both animal isoforms, H and L orplant isoforms (H-1, H-2, etc).

A “ferritin protein subunit” is defined as one of the 12 or 24polypeptide subunits that make up a ferritin protein. The numberingsystem used herein for the identification of amino acids within ferritinsubunits is based on the original sequence of horse spleen L ferritin(Swiss Protein Database Accession Number P02791). The horse spleennumbering system can be easily converted to a numbering system based onthe human H sequence (Swiss Protein Database accession number P02794;the human L sequence accession number is P02792), which has fouradditional amino acids at the N-terminus. The human H sequence numberingtherefore adds 4 to the corresponding amino acid number in horse spleenferritin. For example, L134 by horse spleen numbering corresponds toL138 by human H sequence numbering. Alignments of ferritin subunitsequences can be found, e.g., in Theil, E. C., in Handbook ofMetalloproteins, (Messerschmidt, A. et al., eds.), John Wiley & Sons,Chichester, UK, pp. 771-81, 2001; Waldo, G. S. and Theil, E. C., inComprehensive Supramolecular Chemistry, Vol. 5, (K. S. Suslick, ed.),Pergamon Press, Oxford, UK, pp. 65-89, 1996.

“Apoferritin” is ferritin in the protein cage, i.e., in theunmineralized state.

A “ferritin pore” is one of the external or internal ferritin cage poresthat lead to the eight Fe²⁺ exit/entry ion channels in an assembledferritin protein cage; the channels and pores are formed by trimers offerritin subunits. In an intact, 24 subunit ferritin protein cage, thereare eight three-fold axes of symmetry, each at a junction of threeferritin subunits. Each ferritin pore and ion channel is formed by thesethree-way junctions of ferritin subunits. The pores can be visualized incrystals of ferritin proteins by X-ray crystallography and analyzed insolutions by changes in the rate of Fe²⁺ exit.

An “immunoblot” or “immunodetection” is a specific type of biochemicaltest that measures the presence or concentration of a protein (referredto as the “analyte”) in solutions that frequently contain a complexmixture of substances including other proteins. The methods andtechniques involved in immunoassays are well known by those in the art.

“Isolation” or “isolation of ferritin” as used herein means separationof ferritin from other components in the plant or animal material, whichprovides a substantially pure target compound, such as a substantiallypure ferritin. Substantially pure ferritin contains ferritin in anamount of from about 50% to about 100%, from about 50% to about 80%,from about 70% to about 85%, from about 65% to about 95% by weight ofthe total protein in the material processed by the method of theinvention.

The terms “individual,” “subject” and “patient” are used interchangeablyherein, and generally refer to a mammal. The term “mammal” is defined asan individual belonging to the class Mammalia and includes, withoutlimitation, humans, domestic and farm animals, and zoo, sports, and petanimals, such as cows, sheep, dogs, horses, cats and cows.

A “legume” can be one or more soybeans, yellow peas, green peas,lentils, chickpeas (also called garbanzos), peanuts, trefoil, pintobeans, Great Northern beans, navy beans, red beans, black beans, dark orlight red kidney beans, fava beans, baby lima beans, pink beans,mayocoba beans, small red beans, black-eyed peas (also called cow peas),cranberry beans, white beans, rice beans, butter beans, and combinationsof any of the foregoing. The legume can be any of a variety of species,including, e.g., a Phaseolus species (e.g., Phaseolus vulgaris), a Pisumspecies (e.g., Pisum sativum), a Lens species (e.g., Lens vulgaris, Lensculinaris), a Cicera species (e.g., Cicera arietenum), a Vigna species(e.g., Vigna unguiculata), a Glycine species (e.g., Glycine max), andcombinations of any thereof.

The term “nutraceutical formulation” refers to a food or part of a foodthat offers medical and/or health benefits including prevention ortreatment of disease. Nutraceutical products range from isolatednutrients, dietary supplements and diets, to genetically engineereddesigner foods, functional foods, herbal products and processed foodssuch as cereal, soup and beverages. The term “functional foods,” refersto foods that include “any modified food or food ingredients that mayprovide a health benefit beyond the traditional nutrients it contains.”Nutraceutical formulations of interest include foods for veterinary orhuman use, including food bars (e.g. cereal bars, breakfast bars, energybars, nutritional bars); chewing gums; drinks; fortified drinks; drinksupplements (e.g., powders to be added to a drink); tablets; lozenges;candies; and the like.

The term “solution” refers to a composition comprising a solvent and asolute, and includes true solutions and suspensions. Examples ofsolutions include a solid, liquid or gas dissolved in a liquid andparticulates or micelles suspended in a liquid.

A “supplement” or “dietary supplement” as used herein is useful forsupplementing, replenishing, and increasing the iron supply to humans,animals and plants, and for treating various disorders and conditions. Adietary supplement can be formulated for oral administration. Ascontemplated in the present invention, a dietary supplement includesferritin in an amount of from about 10% to about 90% by weight of thetotal protein in the supplement. For example, subject dietary supplementincludes ferritin in an amount of from about 10% to about 15%, fromabout 15% to about 20%, from about 20% to about 25%, from about 25% toabout 30%, from about 30% to about 35%, from about 35% to about 40%,from about 40% to about 45%, from about 45% to about 50%, or from about55% to about 90% by weight of the total protein in the supplement. Fororal preparations, a subject dietary supplement can be formulated withappropriate additives to make tablets, powders, granules or capsules,for example, with conventional additives, such as lactose, mannitol,corn starch or potato starch; with binders, such as crystallinecellulose, cellulose derivatives, acacia, corn starch or gelatins; withdisintegrators, such as corn starch, potato starch or sodiumcarboxymethylcellulose; with lubricants, such as talc or magnesiumstearate; and if desired, with diluents, buffering agents, moisteningagents, preservatives and flavoring agents. A dietary supplement can beadministered in one, or more than one, doses per day. A dietarysupplement can be administered at various frequencies, e.g., four timesdaily, three times daily, twice daily, once daily, every other day,three times per week, twice per week, or once per week.

A “therapeutic composition” as used herein means a substance that isintended to have a therapeutic effect such as a pharmaceuticalcomposition, a nutraceutical, a dietary supplement, and othersubstances. A therapeutic composition may be configured to contain apharmaceutically acceptable carrier. The therapeutic composition maycontain pharmaceutically acceptable excipients, such as vehicles,adjuvants, carriers or diluents. Moreover, pharmaceutically acceptableauxiliary substances, such as pH adjusting and buffering agents,tonicity adjusting agents, stabilizers, and wetting agents.

As used herein, the phrases “therapeutically effective amount” and“prophylactically effective amount” refer to an amount that provides atherapeutic benefit in the treatment, prevention, or management of adisease or an overt symptom of the disease. The therapeuticallyeffective amount may treat a disease or condition, a symptom of disease,or a predisposition toward a disease, with the purpose to cure, heal,alleviate, relieve, alter, remedy, ameliorate, improve, or affect thedisease, the symptoms of disease, or the predisposition toward disease.The specific amount that is therapeutically effective can be readilydetermined by ordinary medical practitioner, and may vary depending onfactors known in the art, such as, e.g. the type of disease, thepatient's history and age, the stage of disease, and the administrationof other therapeutic agents.

The terms “treatment” or “treating” as used herein covers any treatmentof a condition treatable by iron in a living organism, preferably aprimate, and more preferably a human, and includes:

-   -   (i) preventing the condition from occurring in a subject which        may be predisposed to the condition but has not yet been        diagnosed as having it;    -   (ii) inhibiting the condition, e.g., arresting or slowing its        development; or    -   (iii) relieving the condition, e.g., causing regression of the        condition.

Stated quantities in the specification and claims are intended toencompass variations of the stated amounts consistent with the practiceof the present invention. Such variations are readily determined by oneof skill in the art following procedures outlined in specification andtypically encode variation on the order of +/−10-20%.

Ferritin Iron

Ferritin is a unique form of dietary iron in that it contains a proteincage with caged iron oxide mineral. In contrast with other ironsupplements and dietary iron sources, ferritin's protein coat protects auser's intestine from free radical chemistry caused by iron, which canirritate the intestine. In addition, ferritin iron is released slowlyinto the blood from the intestine, which also allows for protection ofthe intestine from free radical chemistry caused by iron.

Plant ferritin contains an average of 1000 iron atoms per protein cage,and animal ferritin contains an average of 1500-2000 iron atoms perprotein cage, which allows for more efficient absorption of ferritiniron by the body. In other words, for one transport event in theintestine, the user's body would obtain 1000 times as much iron as itwould from non-heme iron salts and chelators.

Ferritin iron is well absorbed by animals. In a rat model, ferritin hasbeen shown to cure iron deficiency. In humans, ferritin iron is absorbedon the order of 20-30%.

Ferritin iron is absorbed by the intestine using a mechanism differentfrom the mechanism of absorption of other iron sources such as non-hemeiron supplements or heme iron from meat. Humans have been consumingferritin iron for millennia in forms such as ferritin-rich legumes,which have been cultivated for 12,000 years. All contemporary culturesinclude legumes in their traditional and modern diets. In addition,plant ferritin can be eaten by vegans, which is important since manyvegan diets are iron deficient and need to be supplemented with iron.

Isolation of Ferritin from Plant Material

Plant material can be used as a starting material to isolate ferritin ina substantially pure form. Ferritin can be isolated from plant materialby the steps of: (a) separation of the plant material into a soluble andinsoluble fraction, (b) removal of non-ferritin components from thesoluble fraction of step (a), thereby isolating ferritin, and (c)concentration of the isolated ferritin from step (b).

This method of ferritin isolation is less labor-intensive and costlythan previously described methods. Previous methods of ferritinextraction from plants include the grinding of plant material in liquidnitrogen, followed by the addition of extraction buffer andclarification by filtration and centrifugation. (Ragland, M. et al., JBiol Chem. 265:18339-44 (1990).) However, such a method is suitable onlyfor research due to the labor-intensive and costly nature of the method.

A typical starting plant material is a legume such as a soybean. Inaddition to soybeans, yellow peas, green peas, lentils, chickpeas,peanuts, trefoil, pinto beans, Great Northern beans, navy beans, redbeans, black beans, dark or light red kidney beans, fava beans, greenbaby lima beans, pink beans, mayocoba beans, small red beans, black-eyedpeas, cranberry beans, white beans, rice beans, butter beans, or acombination thereof can be used as starting plant material.

It is contemplated in the present invention that the plant material usedto isolate ferritin can include the whole plant, or any ferritin-richportion of a plant, e.g., seed, stem, fruit, leaf, root (e.g.,nodulating root), flower, stem, etc. In some cases, the source of theferritin is one or more of a seed, a nodulating root, and a leaf. Wherethe source of the ferritin is a seed or a bean, the ferritin can beobtained from the whole seed or bean, or a part of a seed or bean, e.g.,the hull.

Plant seeds from legumes such as soybeans have a high ferritin contentcompared to other plant seeds; immature and senescent sections of leavesand as nitrogen-fixing nodules as accumulate ferritin. Soybean seedshave been tested and shown to be a good source of iron for animal andhuman nutrition (Murray-Kolb et al, Am J Clin Nutr. 77:180-4, 2003;Davila-Hicks et al. Am J Clin Nutr. 80:936-40, 2004; Theil E. C. et al,J Nutr 142: 478-83, 2012; Beard, J. L. et al., J. Nutr. 126:154-60,1996) and survives human digestion largely intact (Theil E. C. et al.,J. Nutr. 142:478-83, 2012). In cell cultures studies, intact ferritin istransported from the apical side of polarized intestinal cell models viareceptor-mediated endocytosis (San Martin et al., J Nutr. 138:659-66,2008). Iron absorbed by the intestine from intact ferritin is processedintracellularly and released from the basolateral side (Theil E. C. etal., J. Nutr. 142:478-83 2012). Purified ferritin and soybean meal aresources of iron for treating iron deficiency in the rat model system.(Beard, J. L et al., J Nutr. 126:154-60 1996).

The starting plant material can also be a processing stream or a wastestream resulting from the processing of soy or other beans. For example,the source of the isolated ferritin can be a waste stream from theproduction of tofu or soy milk from soybeans. Processing soy for soymilk produces an insoluble by-product of soy, called okara. Either wetor dried okara or other material from legume waste process streams canbe used as starting plant material.

The plant material from the waste stream and/or the legumes themselvesis treated to isolate the ferritin, followed by concentration of theferritin. The concentrated ferritin can then be used to treat humans andother animals in need thereof, such as, for example, treatment of aniron deficiency.

The step of separation of the plant material into a soluble andinsoluble fraction can be done through any separation technique, suchas, for example, mechanical separation.

Non-ferritin components can be removed from the soluble fraction of theseparated plant material by any means, such as, for example, enzymatictreatment of the soluble fraction. The enzyme can be one or more of acarbohydrate degrading enzyme(s). In a preferred embodiment, one or moreof the enzymes used in the method is a glycosidase enzyme.Alternatively, or in addition, one or more of the enzymes employed inthe method can be an amylase, in particular an α-amylase or a β-amylase,an arabinanase, an arabinofuranosidase, a galactanase, anα-galactosidase, a β-galactosidase, a polygalacturonase, a pectin methylesterase, a rhamnogalacturonase, a rhamnogalacturon acetyl esterase, apectin lyase, a xylanase, a cellulase, a β-glucosidase, acellobiohydrolase, a xylosidase, a mannanase, and/or a glucuronisidase.The enzymes are used in a dosage normally employed for degradingcarbohydrates.

After enzymatic treatment, the soluble fraction can be dialyzed toremove the low molecular weight, non-ferritin components. Concentrationof the isolated ferritin can be achieved by any means, such as, forexample, drying the ferritin-containing soluble fraction, or byultrafiltration.

Additional steps in the method of isolating ferritin can includefractionation of the soluble fraction after the non-ferritin componentsare removed. In addition, the method of isolating ferritin can includethe step of treatment of the starting material or soluble fraction withan inorganic or organic solvent. The solvent treatment can occur beforeor after the removal of the non-ferritin components from the solublefraction.

The product of the method of this invention is referred to as isolatedferritin, purified ferritin, or substantially purified ferritin. Theferritin isolated by the method of the invention is useful forindustrial applications, as described further below.

Extraction of Ferritin from Animal Material

In animals, ferritin is present in high amounts in the liver, kidney,spleen, and bone marrow. The present invention contemplates thatferritin may be derived from the tissues of animals. Iron-containingferritin derived from animals can be also used by humans and otheranimals that are in need of increased iron in their diet.

Isolation of ferritin from animal material can be done using the stepsof: (a) separation of the animal material into soluble and insolublefractions; (b) heat-denaturation of the soluble fraction; (c) removal ofcarbohydrates from the heat-denatured soluble fraction by enzymedigestion; and (d) concentration of the enzymatically treated,heat-denatured soluble fraction, thereby isolating ferritin.

The starting animal material can be animal tissues such as liver,kidney, spleen or bone marrow from an animal.

The separation of soluble and insoluble material can be achieved throughany means, such as, for example, mechanical separation.

The step of heat-denaturation can performed at temperatures adequate todenature proteins. It is contemplated that temperatures below 80° C. canbe used.

Carbohydrates can be removed from the heat-denatured soluble fraction byany means, such as, for example, enzymatic treatment of the solublefraction. The enzyme can be one or more of a carbohydrate degradingenzyme(s). In a preferred embodiment, one or more of the enzymes used inthe method is a glycosidase enzyme. Alternatively, or in addition, oneor more of the enzymes employed in the method can be an amylase, inparticular an α-amylase or a β-amylase, an arabinanase, anarabinofuranosidase, a galactanase, an α-galactosidase, aβ-galactosidase, a polygalacturonase, a pectin methyl esterase, arhamnogalacturonase, a rhamnogalacturon acetyl esterase, a pectin lyase,a xylanase, a cellulase, a β-glucosidase, a cellobiohydrolase, axylosidase, a mannanase, and/or a glucuronisidase. The enzymes are usedin a dosage normally employed for degrading carbohydrates. Concentrationof the isolated ferritin can be achieved through any means such as, forexample, drying with low heat, ultrafiltration, or spray drying.

The addition of other steps in the method to isolate ferritin fromanimal material is contemplated. For example, the enzymatically treatedsoluble fraction can be size fractionated to remove the digestedcarbohydrate material. In addition, the method of isolating ferritin caninclude the step of treatment of the soluble fraction with an inorganicor organic solvent. The solvent treatment can occur before or after theremoval of the non-ferritin components from the soluble fraction.

Industrial Applications

The isolated ferritin obtained by the method of the invention fromeither plant or animal starting material may have various industrialapplications. The isolated ferritin is particularly useful for beingadded into products for human or animal nutrition, such as food,beverages, nutraceuticals and supplements.

It is contemplated that the isolated ferritin obtained by the methodsdescribed herein can be added to a heat-processed substance to increasethe iron content of the heat-processed substance. A heat-processedsubstance can be food or a beverage. The isolated ferritin can be addedto the heat-processed substance either before or after the substance isheat-processed.

Treatment or Prevention of Iron Deficiency

The methods of the present invention contemplate that a therapeutic orsupplementary composition containing the plant or animal ferritinisolated by the methods described above can be delivered to an organismin need of iron by any known means. The organism can be an animal suchas a mammal, including a human. Alternatively, the organism can be aplant. In a preferred embodiment, the composition can be deliveredorally.

The ferritin containing therapeutic or supplemental compositions of theinvention can be administered in dosages sufficient to treat or preventiron deficiency. A therapeutic or prophylactic amount effective to treata disorder related to iron deficiency (such as, for example, anemia) bythe methods disclosed herein comprises a sufficient amount of the plantor animal derived ferritin agent or therapeutic composition containingplant or animal derived ferritin delivered during the entire course oftreatment to ameliorate or reduce the symptoms of the disorder beingtargeted for treatment. The composition can also contain apharmaceutically acceptable carrier or excipient. Such carriers orexcipients include any pharmaceutical agent that does not itself inducethe production of antibodies harmful to the individual receiving thecomposition, and which may be administered without undue toxicity.

The isolated ferritin agents and therapeutic compositions can beadministered by continuous delivery, intermittent delivery, or through acombination of continuous and intermittent delivery. Many factors caninfluence the dosage and timing required to effectively treat a subject,including, but not limited to, the severity of the disease or disorder,previous treatments, the general health and/or age of the subject, andother diseases present. Moreover, treatment of a subject with atherapeutically effective amount of a composition can include a singletreatment or a series of treatments. In addition to their administrationindividually or as a plurality, the therapeutic compositions of theinvention can be administered in combination with other known agentseffective in treatment of diseases. In any event, the administeringphysician can adjust the amount and timing of administration of thetherapeutic composition on the basis of results observed using standardmeasures of efficacy known in the art or described herein.

Quantitation of Ferritin Iron

Rough determination of the ferritin protein and iron concentration inplant extracts has been shown previously using quantitative Westernblotting with a rabbit polyclonal anti-soybean ferritin antiserum.(Ragland, M. and Theil, E. C., Plant Mol. Biol. 21:555-560, 1993;Kimata, Y. and Theil, E. C., Plant Physiol. 104:263-270, 1994; Burton,J. W., et al., J. Plant Nutr. 21:913-927, 1998)

The methods of the present invention contemplate an accuratequantitation of iron derived from plant and animal ferritin, eithernatural or from recombinant apoferritin with reconstituted iron mineralusing either classical, quantitative Western blots or automated,quantitative immunoanalysis using capillary electrophoresis incombination with determination of the amount of ferritin iron andquantitation of the total protein in the sample. The iron can becontained in food, dietary supplements, nutriceuticals and the like.Ferritin iron derived from any natural or processed material can bequantitated, including ferritin isolated by the procedures describedherein.

In order to determine the amount of ferritin iron in a sample, thesample's weight percentage of a ferritin iron source must be known. Theferritin iron source may be from plant or animal material. The samplecan be any type of sample, for example, natural or processed materialincluding samples from food, beverage, plant or animal material. Inaddition, the amount of ferritin iron in tissue derived from animals orhumans can be determined, including the normal tissue orhyperferritinemic serum of a subject.

The steps for determining the amount of ferritin iron in a sample wherethe sample has a known weight percentage of a ferritin iron sourceinclude the steps of: (a) measuring the total iron in the sample perunit weight of the sample; (b) preparing a soluble extract of a knownweight per volume of the sample from step (a) to make a soluble sampleextract, (c) determining the total iron per unit weight of the solublesample extract of the sample; (d) determining the ferritin protein perunit weight in the soluble sample extract, using quantitativeimmunological analysis; and (e) calculating the amount of ferritin ironin the sample using the amount of the total iron from step (c) dividedby the amount of ferritin protein from step (d), multiplied by theamount of total iron in the sample from step (a) multiplied by acorrection factor, thereby determining the amount of ferritin iron inthe sample. The equation is as follows: a(c/d)(correction factor)=e.

The correction factor used in the calculation depends on the source ofthe ferritin iron in the sample. For example, the correction factor is0.75 for a sample comprising legumes, which would make the equationabove as follows: a(c/d)(0.75)=e.

The quantitative immunological analysis used in step (d) described abovecan be any type of quantitative immunological analysis such as, forexample, a quantitative immunoblot, Western blot analysis, orquantitative capillary immunoelectrophoresis.

It is also contemplated that the components required to determine theamount of ferritin iron in a sample can be provided in a kit.

EXAMPLES Example 1 Isolation of Ferritin from Plants

In order to isolate ferritin from plants, the starting plant materialwas separated into soluble and insoluble fractions after adding aneutral, aqueous salt solution buffered at near neutrality. The mixturecan be chilled, at room temperature, or heated. In this example, theplant material/salt solution was kept chilled at between 32 and 43degrees Fahrenheit (0 to 6 degrees Celsius) overnight with slow stiflingor agitation. More extreme conditions of temperature, pH and solvent,which do not interfere with the immunoanalysis, might also be used.Next, the plant material/saline extract was filtered, followed byclarification using continuous or static centrifugation at greater than35,000×g for 20 minutes.

Carbohydrate was partially removed from the soluble fraction obtainedfrom the clarification step. Carbohydrate was removed by a mixture ofglycosidase enzymes added either during extraction or afterclarification. FIG. 1 shows the removal of a carbohydrate, namely starchor cellulose, in ferritin extracts that were treated with a glycosidase.The graph demonstrates the conversion of carbohydrate inglycosidase-treated ferritin extract to reducing sugars at roomtemperature over time. In this example, released sugar was measured asthe absorbance at 500 nm of the 3,5 dinitrosalicylic acid-sugar complexand analyzed by UV-vis spectrophotometry.

After the carbohydrates were removed enzymatically, the clarifiedsoluble solution was dialysed with size selective membranes in order toremove the digested sugars and enzyme. The fractionated soluble solutionwas then dried using low heat to concentrate the isolated ferritin.

The resulting preparation containing isolated ferritin is then used as afood or beverage additive, or as a supplement to treat humans and otheranimals who are iron deficient. To date, concentrations of ferritin ofmore than 1000-fold have been achieved by this process.

Example 2 Ferritin Isolation from Animals

Ferritin can be isolated from animal material such as, for example,liver, kidney, spleen, bone marrow, or other ferritin-rich source.Isolation of ferritin from animal material can be done using the stepsof: (a) separation of the animal material into soluble and insolublefractions; (b) heat-denaturation of the soluble fraction; (d) removal ofcarbohydrates from the heat-denatured soluble fraction by enzymedigestion; and (c) concentration of the enzymatically treated,heat-denatured soluble fraction. Iron-containing, isolated ferritinderived from animals can be also used by humans and other animals thatare in need of increased iron in their diet.

Example 3 Determination of Total Ferritin Content

The ferritin content can be determined from any substance containingferritin including a processed substance (i.e., food, supplement,nutriceutical) derived from animal or plant starting material. In thisexample, the ferritin iron was quantitated in a food sample containingferritin iron from legumes.

First, the total food iron content was determined. Next, the ironconcentration, the percentage amount of the non-legume component and thepercentage amount of the legume component of the processed substance wasdetermined. From the resulting numbers, the legume iron content of thetotal processed substrate was calculated.

The amount of iron per ferritin protein was then determined and for theprotein fraction of the extract. To do this, an aqueous extract of theprocessed substance was prepared using, as an example, 3 milliliters(mls) of extracting material per milligram (mg) of the processedsubstance. The total iron concentration in the extract is determined.The concentration of specific ferritin protein in the extract is alsodetermined, using a quantitative immunological analysis, e.g.quantitative immunoblot or automated capillary electrophoresis coupledto quantitation of chemiluminesence or fluorescence with an antiserumdirected towards whole legume ferritin or to a ferritin peptide, Extractprotein concentrations are determined with a standard protein assay,such as the Bradford colorimetric protein analysis, which is based onprotein binding of a dye such as Coomassie blue. The dry weight of asample aliquot is determined. The amount of iron per ferritin protein,the amount of ferritin iron and the amount of total legume protein/ml ofextract and /gm of legume starting material is calculated from thecombined results of the iron, protein and ferritin-specificimmunoanalyses. The maximum and minimum ferritin content of processedfood is determined. The amount legume ferritin iron per gram of food iscalculated from the grams of legume added per gram of food and the gramsof legume ferritin iron/gm of legume added to the processed food. Theminimum amount of ferritin per gram of processed food is calculated bymultiplying the resulting number by 0.75 to account for the maximumamounts of nonferritin iron in legumes. If animal ferritin were thesource of the ferritin, measurement of heme iron and the correspondingcorrection to the total iron determination would be made.

In addition to ferritin measured in natural and processed material, theamount of iron-containing ferritin in a tissue, or in serum duringhyperferritinemia in animals or humans can be determined by use of aspecific antiserum directed towards the ferritin and quantitativeimmunoanalyses such as Western blotting or automated capillaryelectrophoresis.

Although the present invention has been described in considerable detailwith reference to certain preferred embodiments, other embodiments arepossible. The steps disclosed for the present methods, for example, arenot intended to be limiting nor are they intended to indicate that eachstep is necessarily essential to the method, but instead are exemplarysteps only. Therefore, the scope of the appended claims should not belimited to the description of preferred embodiments contained in thisdisclosure. All references cited herein are incorporated by reference intheir entirety.

What is claimed is:
 1. A dietary supplement, the dietary supplementcomprising plant ferritin isolated from plant material by the methodcomprising: (a) separation of the plant material into a soluble andinsoluble fraction; and (b) removal of enzymatically digestiblenon-ferritin components from the soluble fraction by treatment of thesoluble fraction with one or more glycosidase enzymes.
 2. The dietarysupplement of claim 1, wherein the plant material is a legume.
 3. Thedietary supplement of claim 1, wherein the plant material comprises thewaste stream from the processing of legumes.
 4. The dietary supplementof claim 1, wherein the isolated plant ferritin comprises 10-50% byweight of the total protein in the dietary supplement.
 5. The dietarysupplement of claim 1, wherein the dietary supplement further comprisesat least one carrier.
 6. The dietary supplement of claim 1, wherein thedietary supplement further comprises at least one excipient.
 7. Adietary supplement, the dietary supplement comprising isolated plantferritin, the dietary supplement comprising plant ferritin isolated fromplant material by the method comprising: (a) separation of the plantmaterial into a soluble and insoluble fraction; and (b) removal ofnon-ferritin components from the soluble fraction by enzymatic treatmentof the soluble fraction with one or more carbohydrate-degrading enzymes,thereby isolating ferritin.
 8. The dietary supplement of claim 7,wherein the plant material is a legume.
 9. The dietary supplement ofclaim 7, wherein the plant material comprises the waste stream from theprocessing of legumes.
 10. The dietary supplement of claim 7, whereinthe isolated plant ferritin comprises 10-50% by weight of the totalprotein in the dietary supplement.
 11. The dietary supplement of claim7, wherein the dietary supplement further comprises at least onecarrier.
 12. The dietary supplement of claim 7, wherein the dietarysupplement further comprises at least one excipient.
 13. A dietarysupplement, the dietary supplement comprising isolated plant ferritin,the dietary supplement comprising plant ferritin isolated from plantmaterial by the method comprising: (a) separation of the plant materialinto a soluble and insoluble fraction; and (b) removal of non-ferritincomponents from the soluble fraction by enzymatic treatment of thesoluble fraction, thereby isolating ferritin.
 14. The dietary supplementof claim 13, wherein the plant material is a legume.
 15. The dietarysupplement of claim 13, wherein the plant material comprises the wastestream from the processing of legumes.
 16. The dietary supplement ofclaim 13, wherein the isolated plant ferritin comprises 10-50% by weightof the total protein in the dietary supplement.
 17. A nutraceuticalproduct, the nutraceutical product comprising isolated plant ferritin,the nutraceutical product comprising plant ferritin isolated from plantmaterial by the method comprising: (a) separation of the plant materialinto a soluble and insoluble fraction; and (b) removal of non-ferritincomponents from the soluble fraction by enzymatic treatment of thesoluble fraction, thereby isolating ferritin.
 18. The nutraceuticalproduct of claim 17, wherein the nutraceutical product isheat-processed.
 19. The nutraceutical product of claim 18, wherein theisolated plant ferritin is added to the nutraceutical product before itis heat-processed.
 20. The nutraceutical product of claim 18, whereinthe isolated plant ferritin is added to the nutraceutical product afterit is heat-processed.